胆红素氧化酶凝胶电泳活性染色法

Active-staining of Bilirubin Oxidase After PAGE

胆红素氧化酶能够特异地将胆红素氧化成胆绿素,利用酶与底物的这种特殊关系可以在凝胶上进行底物染色来直接显示酶的活性区带,这种方法称为胆红素氧化酶的活性染色法.这种活性染色可以采用胆红素直接溶入分离胶后电泳(胆红素-PAGE),也可以在电泳后置于胆红素溶液中染色(PACE-胆红素).其中PAGE-胆红素方法在20 min后即在凝胶上出现深绿色的胆红素氧化酶条带,随着时间延长,凝胶上的绿色酶条带逐渐扩散,最终形成透明条带;而运用胆红素-PAGE的方法则在12 h后才能在凝胶上看见透明的胆红素氧化酶所在的条带.相比之下,前者更为简便、快速、灵敏.

The active-band of bilirubin oxidase after PAGE could be detected easily based on oxidation of the bilirubin into biliverdin by the enzyme, and the band became either green or transparent. The active-staining method was named as the PAGE-bilirubin, the operation of which was the same with the usual PAGE, the only difference was to stain the gel in the bilirubin solution for 20 minutes after the electrophoresis and the green bands of the bilirubin oxidase could be observed on the gel. With the time prolong, the green bands of the biliribin oxidase on the gel gradually became transparent. The staining method could be processed by adding bilirubin into polyacrylamide gel（bilirubin-PAGE） too, the transparent bands of the bilirubin oxidase on the gel could also be observed, but the gel need to be placed in the darkroom for 12 hours. Compare PAGE-bilirubin and bilirubin-PAGE, the PAGE-bilirubin active-staining method is more convenient, fast and sensitive.